The present invention relates to nucleic acid and amino acid sequences of a novel human pancreatitis-associated (PAP) protein, which comprises a soluble C-type lectin. This novel human PAP protein shares features with other proteins in the reg/PSP multigene family which are involved in the regulation of cell growth. The present invention relates to the use of these novel sequences in the diagnosis, prevention and treatment of disease.
Lectins are proteins which are defined by their ability to bind carbohydrates specifically and to agglutinate cells. Lectins have been shown to be involved in a wide variety of cellular functions including cell-cell and cell-matrix interactions. Lectins are widespread among plants, invertebrates and mammals.
Animal lectins have been grouped into four distinct families: 1) C-type lectins, which include selecting; 2) P-type lectins; 3) galectins (formerly termed S-type lectins or S-Lac lectins); and 4) pentraxins [Barondes SH et al. (1994) J. Biol. Chem. 269:20807-10]. The C-type lectins bind carbohydrate ligands in a Ca2+-dependent manner and are structurally related to the asialoglycoprotein receptor. Selectins, a subcategory of the C-type lectins, are composite transmembrane molecules which are involved in cell-cell interactions. The selectins include lymphocyte homing receptors and platelet/endothelial cell surface receptors [Stoolman (1989) Cell 56:907-10].
C-type animal lectins contain Ca2+-dependent carbohydrate-recognition domains (CRDs). The prototypical C-type animal lectins are integral membrane proteins (e.g., the asialoglycoprotein receptor); however, a number of soluble C-type animal lectins have been identified. One group of soluble C-type animal lectins, termed collections or Group III C-type lectins, comprise proteins having both lectin- (i.e., CRD) and collagenous-like domains within a single polypeptide [Drickamer (1993) Curr. Opin. Struct. Biol. 3:393]. Another group of soluble C-type animal lectins, termed Group IV C-type lectins, comprise free CRDs which are not joined to other polypeptide domains (other than a signal peptide utilized in secretion) [Drickamer (1993), supra]. The soluble C-type animal lectins comprising free CRDs found in mammals are most closely related to proteins identified in invertebrates and lower vertebrates (e.g., snakes).
Proteins recognized as members of the Group IV C-type lectins appear to be members of a multigene family termed the reg/PSP multigene family [Drickamer (1993), supra and Unno et al. (1993) J. Biol. Chem. 268:15974]. The reg/PSP multigene family comprises genes encoding secretory proteins which are expressed in the pancreas; the ectopic expression (i.e., expression in a tissue which does not normally express reg/PSP proteins) of some members of the reg/PSP family is associated with disease states such as tumors and Alzheimer""s disease.
The first member of the reg/PSP multigene family was identified in a cDNA library derived from rat regenerating pancreatic islets [Terazono et al. (1988) J. Biol. Chem. 263:2111]. This gene was termed reg (regenerating gene) and is now known as the regIxcex1 gene. The regIxcex1 gene product has been called by different investigators reg protein, regIxcex1 protein, lithostathine, islet cell regeneration factor (ICRF), pancreatic stone protein (PSP) and pancreatic thread protein (PTP) [Terazono et al. (1988), supra; Moriizumi et al. (1994) Biochem. Biophys. Acta 1217:199; Dusetti et al. (1993) Biochem. Biophys. Acta 1174:99; Rouquier et al. (1991) J. Biol. Chem. 266:786; and de la Monte et al. (1990) J. Clin. Invest. 86:1004]. The clear association between reg gene expression and islet cell replication in vitro has lead to the suggestion that the regIxcex1/lithostathine protein has a growth-promoting activity for islet xcex2-cells [Unno et al. (1993), supra]. Human regIxcex1 mRNA is expressed in colon and rectal tumors although it is not expressed in normal colon or rectal tissue. Thus, ectopic expression of regIxcex1 protein is associated with tumorigenesis. Elevated levels of regIxcex1 protein have been found in the brains of patients suffering from Alzheimer""s disease as well as in the brains of middle-aged individuals with Down""s syndrome [Ozturk et al. (1989) Proc. Natl. Acad. Sci. USA 86:419 and de la Monte et al. (1990) J. Clin. Invest. 86:1004]. RegIxcex1 mRNA is expressed in the developing human brain, but not in normal adult brain; expression of regIxcex1 is seen in adult brain which is undergoing regenerative sprouting. Given its pattern of expression (e.g., expression in regenerating pancreatic islets and brain, expression in tumors), it appears that regIxcex1 protein is associated with cell growth.
Other members of the reg/PSP multigene family are the genes encoding pancreatitis-associated proteins (PAPs) which have been identified in humans, mice and rats [Iovanna et al. (1991) J. Biol. Chem. 266:24664; Orelle et al. (1992) J. Clin. Invest. 90:2284; Itoh and Teraoka (1993) Biochem. Biophys. Acta 1172:184; and Dusetti et al. (1994) Genomics 19:108]. The reg/lithostathine and PAP proteins characterized to date share about 45-65% identity on the amino acid level.
The PAP proteins are secretory proteins which are stored in zymogen granules prior to secretion [Keim et al. (1991) Gastroenterol. 100:775]; PAP is present at low levels in normal pancreas but is rapidly overexpressed during the acute phase of pancreatitis. PAP, like other members of the reg/PSP family, shares sequence similarity with the carbohydrate-binding domain of C-type lectins which likely explains the ability of PAP to induce aggregation of bacteria [Iovanna et al. (1991), supra]. The ability to aggregate bacteria has lead to the suggestion that PAP is involved in the control of bacterial proliferation, a frequent complication of pancreatitis. PAP has been shown to be able to bind lactose [Christa et al. (1994) FEBS Lett. 337:114].
Three PAP genes, PAP I-III, have been identified in rats. All three PAP genes are expressed during the acute phase of pancreatitis. Rat PAP I and PAP III are expressed constitutively in the intestine and their expression is induced by feeding. Rat PAP II is not expressed in the intestine. Rat PAP I and PAP III share 66% amino acid identity; rat PAP II and PAP III share 63% amino acid identity; rat PAP I and PAP II share 58% amino acid identity. A homologue of rat PAP I has been identified in cows [BPTP; de la Monte et al. (1990), supra].
A human homolog of the rat PAP I gene, human PAP or human PAP I, has been identified [Orelle et al. (1992) J. Clin. Invest. 90:2284]. The human PAP I protein is the same size as the rat PAP I protein (175 amino acids) and these two proteins share 71% amino acid identity, including conservation of 7 cystine residues. Both the rat and the human PAP I proteins are synthesized as preproteins having an N-terminal signal peptide of 26 amino acids. Expression of the human PAP I mRNA is increased in necrohemorragic pancreatitis. Serum levels of human PAP I were found to be near background levels in normal individuals; in individuals suffering from acute pancreatitis or acute exacerbations of chronic pancreatitis, human PAP I levels increased 24-140 times the background level [Orelle et al. (1992), supra]. Thus, human PAP I appears to serve as a marker of acute pancreatitis.
The human PAP I gene is also referred to as the HIP gene [Lasserre et al. (1992) Cancer Res. 52:5089]. The HIP gene was identified by differential screening of a human primary liver cancer (hepatocellular carcinoma) library. The human PAP I/HIP gene is not expressed in normal adult or fetal liver; expression of PAP I/HIP is limited to the pancreas and small intestine in normal tissues. Thus, the ectopic expression of PAP I/HIP is associated with tumorigenesis in the liver. In addition, PAP I/HIP mRNA is expressed in human pancreatic cell hyperplasia (hyperinsulinism) (Lasserre et al., supra).
Proteins expressed by the reg/PSP multigene family represent an important family of proteins which are involved in the maintenance of proper pancreatic function as well as in the regulation of cell proliferation and/or differentiation. Discovery of new molecules related to or in the mammalian reg/PSP multigene family is useful for the development of new diagnostic or therapeutic compositions.
The present invention features a novel Group IV C-type lectin protein hereinafter designated human PAP-2 and characterized as having similarity to the human PAP 1 protein. Human PAP-2 is a member of the PAP branch of the reg/PSP multigene family.
Accordingly, the invention features a substantially purified polypeptide having the amino acid sequence shown in SEQ ID NO:1 or fragments thereof. Preferred fragments of SEQ ID NO:1 are fragments which retain biological activity or immunological activity (i.e., capable of eliciting anti-human PAP-2 antibodies). Fragments of SEQ ID NO:1 which are at least 15 amino acids, at least 50 amino acids, at least 100 amino acids, at least 125 amino acids and at least 200 amino acids in length are contemplated. The invention specifically contemplates secretory (i.e., the signal peptide is cleaved; E27-D175 of SEQ ID NO:1) and nonsecretory (i.e., signal peptide remains) forms of a substantially purified human PAP-2 as well as any proteolytic fragments thereof.
The present invention further provides isolated and substantially purified polynucleotide sequences encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2 or variants thereof. In another embodiment, the present invention provides polynucleotides comprising fragments of SEQ ID NO:2 having a length of at least seven nucleotides. The invention further contemplates fragments of this polynucleotide sequence (i.e., SEQ ID NO:2) that are at least 50 nucleotides, at least 100 nucleotides, at least 250 nucleotides, at least 500 nucleotides and at least 750 nucleotides in length.
In addition, the invention provides polynucleotide sequences which hybridize under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. In another embodiment the present invention provides a composition comprising an isolated and purified polynucleotide sequence encoding human PAP-2.
The invention provides polynucleotide sequences comprising the complement of SEQ ID NO:2 or variants thereof; these complementary nucleic acid sequences may comprise the complement of the entire nucleic acid sequence of SEQ ID NO:2 or fragments thereof. In another embodiment the present invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof.
The invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode human PAP-2.
In another embodiment the present invention provides an isolated polynucleotide comprising at least a portion of the nucleic acid sequence of SEQ ID NO:2 or variants thereof contained on a recombinant expression vector. In yet another embodiment, the expression vector containing the polynucleotide sequence is contained within a host cell. The invention is not limited by the nature of the host cell employed. For example, the host cell may be an E. coli cell, a yeast cell, an insect cell, a mammalian cell, etc.
The present invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing an isolated polynucleotide encoding at least a fragment of the human PAP-2 polypeptide under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
In another embodiment, the invention provides a pharmaceutical composition comprising a substantially purified human PAP-2 protein having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antibody which binds specifically to a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist which specifically binds to and modulates the activity of a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:1. The present invention further provides a pharmaceutical composition comprising a purified agonist which specifically binds to and modulates the activity of a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:1. In another embodiment, the invention provides a purified antagonist which specifically binds to and modulates the activity of a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:1. The present invention further provides a pharmaceutical composition comprising a purified antagonist which specifically binds to and modulates the activity of a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:1.
The invention also provides a method for treating pancreatitis (particularly for preventing or controlling bacterial infections associated with pancreatitis) comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a purified agonist which specifically binds to and modulates the activity of a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:1. The treatment of a variety of tumors, including but not limited to tumors of the pancreas, esophagus lung and large intestine (e.g., cecum and colon), using agonists as well as antagonists of human PAP-2 is also contemplated by the present invention.
The invention also provides a method for detection of polynucleotides encoding human PAP-2 in a biological sample comprising the steps of: a) hybridizing a polynucleotide sequence encoding human PAP-2 (SEQ ID NO:1) to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding human PAP-2 in the biological sample. In a preferred embodiment, prior to hybridization, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.